Purpose: To learn how to quantify DNA using the “nanodrop”, how to set up and run a PCR reaction, and how to make an agarose gel

Materials:

• agarose
• gel electrophoresis tub
• TAE
• microwave
• cool bath
• bromophenol blue
• flask
• bleach
• Nanodrop

PCR workspace set up

• Clean your desk with 10% bleach.
• Wear gloves.
• Clean/wipe down your pipettes before use.
• Use autoclaved tubes and filter tips.
• Keep all pipette tip boxes closed when not in use.
• Keep all tubes closed when not in use.
• Minimize the movement/ air flow around your desk.

PCR Reaction Set Up

Perform PCR assay in a total of 25 µL:

1. Add 12.5 µL  2X Master Mix to a flask. Add 0.6 grams of agarose to the solution and dissolve the substance at 45C for annealing.

2. Add 0.6 grams of agarose to the solution and dissolve the substance at 45C for annealing. (the mixture needs to be 1.5% agarose)

3. Allow the solution to cool before inserting 5ul of bromphenol blue in the agarose solution

4. Pour the agarose solution into the electrophoresis tub and allow it to sit still in order to solidify. The next step will be to insert the DNA into the gel.

Conclusion

The lab went smoothly however, I am still a little worried about the amount of DNA that will be present in our sample due to the mishap in the previous lab. After the DNA is placed in the gel, it will move towards the positive side of the tub since DNA is negatively charged. The strands will have to be observed under a UV light, this was the point in adding the bromothenol blue so the DNA will be clearly visible.